Mitochondrial fatty acid beta-oxidation is the major source of energy in highly oxidative mammalian tissues and is essential for intermediary metabolism and production of ketone bodies in liver. Human genetic defects in fatty acid oxidation genes cause acute liver failure, a Reye's syndrome-like phenotype of hypoketotic, hypoglycemia; cardiomyopathy; skeletal myopathy; or sudden, unexpected childhood death. This pathway consists of four enzymatic steps catalyzed by 12 different nuclearly-encoded enzymes that are developmentally-regulated and tissue- specifically expressed. This proposal focuses on two enzymes catalyzing the third step, the short chain 3-hydroxy-acyl-CoA dehydrogenases (SCHAD). Aims 1 and 2 will examine the hypothesis that tissue-specific and developmental expression of the SCHAD-1 and -2 genes is coordinated with other fatty acid oxidation enzyme genes and involves nuclear hormone receptor transcription factors responsive to energy requirements and nutritional cues. Aim 2 will use the human SCHAD-1 gene and in vitro transfection and transgenic mice to locate critical regulatory sequences. Based upon the SCHAD-1 crystal structure and protein homologies, Aim 3 will determine structure-function relationships of SCHAD-1 of normal and mutant SCHADs after expression in bacteria to explore the hypothesis that SCHAD-1 and long chain 3-hydroxy- acyl-CoA dehydrogenase share common structural domains. Using the human gene sequences we have determined, Aim 4 will delineate SCHAD-1 or -2 mutations in children to examine the proposal that SCHAD deficiency and environmental stresses cause a Reye's syndrome-like phenotype of acute hepatic failure. Aim 5 will examine the effects of the three human SCHAD-1 mutations we have discovered on structure and function, the pathogenetics of SCHAD deficiency, to explore the hypothesis that SCHAD missense mutations result in rapid intramitochondrial degradation secondary to misfolding. Aim 6 is to define the phenotype of SCHAD-1 gene ablation we created in mice as a model of human disease, to examine the effects of environmental and drug (aspirin and acetaminophen) stressors on outcome and to similarly study an SCHAD-2 knockout. These studies will augment understanding of the pathogenesis of fatty acid oxidation disorders causing acute liver failure, cardiomyopathy, and sudden death in children.